The invention relates to cellular immunology.
Dendritic cells (xe2x80x9cDCxe2x80x9ds) are potent antigen-presenting cells (xe2x80x9cAPCxe2x80x9ds) in the immune system. It has been shown that DCs provide all the signals required for T cell activation and proliferation. These signals can be categorized into two types. The first type, which gives specificity to the immune response, is mediated through interaction between the T-cell receptor/CD3 (xe2x80x9cTCR/CD3xe2x80x9d) complex and an antigenic peptide presented by a major histocompatibility complex (xe2x80x9cMHCxe2x80x9d) class I or II protein on the surface of APCs. This interaction is necessary, but not sufficient, for T cell activation to occur. In fact, without the second type of signals, the first type of signals can result in T cell anergy. The second type of signals, called costimulatory signals, is neither antigen-specific nor MHC-restricted, and can lead to a full proliferation response of T cells and induction of T cell effector functions in the presence of the first type of signals.
Costimulatory signals are generated by interaction between receptor-ligand pairs expressed on the surface of APCs and T cells. One exemplary receptor-ligand pair is one of the B7 costimulatory molecules on the surface of DCs and its counter-receptor CD28 or CTLA-4 on T cells (Freeman et al., Science 262: 909-911, 1993; Young et al., J. Clin. Invest. 90: 229, 1992; Nabavi et al., Nature 360: 266, 1992).
DCs are minor constituents of various immune organs such as spleen, thymus, lymph node, epidermis, and peripheral blood. For instance, DCs represent merely about 1% of crude spleen (Steinman et al., J. Exp. Med. 149: 1, 1979) or epidermal cell suspensions (Schuler et al., J. Exp. Med. 161: 526, 1985; and Romani et al., J. Invest. Dermatol. 93: 600, 1989), and 0.1-1% of mononuclear cells in peripheral blood (Freudenthal et al., Proc. Natl. Acad. Sci. USA 87: 7698, 1990). Methods for generating dendritic cells from peripheral blood or bone marrow progenitors have been described (Inaba et al., J. Exp. Med. 175: 1157, 1992; Inaba et al., J. Exp. Med. 176: 1693-1702, 1992; Romani et al., J. Exp. Med. 180: 83-93, 1994; and Sallusto et al., J. Exp. Med. 179: 1109-1118, 1994).
The invention features compositions for stimulating an immune system. These compositions each contain a plurality of cells at least half of which (e.g., more than 70-80%) are fused cells, each of which fused cells is generated by fusion between at least one mammalian dendritic cell (e.g., a DC derived from a bone marrow culture or a peripheral blood cell culture) and at least one mammalian non-dendritic cell (e.g., a cancer cell or a transfected cell) that expresses a cell-surface antigen (e.g., a cancer antigen). By xe2x80x9ccancer antigenxe2x80x9d is meant an antigenic molecule that is expressed primarily or entirely by cancer cells, as opposed to normal cells in an individual bearing the cancer. At least half (e.g., at least 70%, and preferably at least 80%) of the fused cells in the compositions express, in an amount effective to stimulate an immune system (e.g., to activate T cells), MHC class II molecules, B7, and the cell-surface antigen. By xe2x80x9cB7xe2x80x9d is meant any member (e.g., B7-1 or B7-2) of the B7 family of costimulatory molecules.
The parental cells used to generate the fused cells can be obtained from a single individual (e.g., a human, a mouse, or a rat). They can also be obtained from different individuals of the same species (e.g., homo sapiens), with matching or non-matching MHC molecules.
Also embraced by the invention are methods of producing fused cells. In these methods, mammalian dendritic cells are fused with mammalian non-dendritic cells expressing a cell-surface antigen in the presence of a fusion agent (e.g., polyethylene glycol or Sendai virus). After culturing the post-fusion cell mixture in a medium (which optionally contains hypoxanthine, aminopterin and thymidine) for a period of time (e.g., 5-12 days), the cultured fused cells are separated from unfused parental non-dendritic cells, based on the different adherence properties of the two cell groups. The unfused parental dendritic cells do not proliferate, and so die off. Even if they remain present in the therapeutic composition, they will not interfere with the effects of the fused cells. The isolated fused cells, which typically express (a) MHC class II protein, (b) B7, and (c) the cell-surface antigen on the non-dendritic parental cells, are useful for stimulating an immune system.
The invention also provides methods of maintaining the DC phenotype of a fused cell by re-fusing it with at least one additional mammalian dendritic cell. The re-fused cells express MHC class II molecules, B7, and the cell-surface antigen of the dendritic parental cells, and are useful for stimulating an immune system.
The compositions of the invention can be administered to an individual (e.g., a human) to stimulate the individual""s immune system. This individual may need an immune stimulation due to infection, or susceptibility to infection, with an intracellular pathogen; cancer; or predisposition to develop cancer. The DCs used to generate fused cells can be obtained from this individual. If this individual has cancer, the individual""s own cancer cells can be used for fusion with his or her own DCs to generate fused cells, which are then administered to the individual.
Stimulation of the immune system with DC fusion cells results in enhancement of the individual""s immunity against a disease condition characterized by abnormal expression of a cell surface antigen that is also present on the parental non-dendritic partners of the fusion cells. Abnormal expression means that the cell surface antigen (i) is not expressed in normal tissue, (ii) is expressed at a much higher level in diseased cells of a given tissue type than in normal tissue of the same type, or (iii) is modified (e.g., phosphorylated) differently in diseased cells of a given tissue type than in normal cells of the same type. Enhancement of immunity entails enhancement of the immune system""s cell-mediated or humoral functions, or both.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting.